APOA5 deficiency causes hypertriglyceridemia by reducing amounts of lipoprotein lipase in capillaries

Apolipoprotein AV (APOA5) deficiency causes hypertriglyceridemia in mice and humans. For years, the cause remained a mystery, but the mechanisms have now come into focus. Here, we review progress in defining APOA5’s function in plasma triglyceride metabolism. Biochemical studies revealed that APOA5 binds to the angiopoietin-like protein 3/8 complex (ANGPTL3/8) and suppresses its ability to inhibit the activity of lipoprotein lipase (LPL). Thus, APOA5 deficiency is accompanied by increased ANGPTL3/8 activity and lower levels of LPL activity. APOA5 deficiency also reduces amounts of LPL in capillaries of oxidative tissues (e.g., heart, brown adipose tissue). Cell culture experiments revealed the likely explanation: ANGPTL3/8 detaches LPL from its binding sites on the surface of cells, and that effect is blocked by APOA5. Both the low intracapillary LPL levels and the high plasma triglyceride levels in Apoa5−/− mice are normalized by recombinant APOA5. Carboxyl-terminal sequences in APOA5 are crucial for its function; a mutant APOA5 lacking 40-carboxyl-terminal residues cannot bind to ANGPTL3/8 and lacks the ability to change intracapillary LPL levels or plasma triglyceride levels in Apoa5−/− mice. Also, an antibody against the last 26 amino acids of APOA5 reduces intracapillary LPL levels and increases plasma triglyceride levels in wild-type mice. An inhibitory ANGPTL3/8-specific antibody functions as an APOA5-mimetic reagent, increasing intracapillary LPL levels and lowering plasma triglyceride levels in both Apoa5−/− and wild-type mice. That antibody is a potentially attractive strategy for treating elevated plasma lipid levels in human patients.

) and transgenic mice that overexpressed human APOA5 (hAPOA5-Tg).Plasma triglyceride (TG) levels were fourfold higher in Apoa5 −/− mice than in Apoa5 +/+ mice and were 65% lower in hAPOA5-Tg mice than in the control mice (1).Subsequent studies revealed that loss-of-function mutations in human APOA5 are associated with high plasma TG levels (2-7) and an increased risk of atherosclerotic coronary artery disease (2,8,9).
The impressive effects of APOA5 on plasma TG levels prompted efforts to define the function of APOA5 in plasma lipid metabolism.Turnover studies involving radiolabeled TG-rich lipoproteins (TRLs) revealed that APOA5 promotes the clearance of TRLs (10)(11)(12)(13), but for years the underlying mechanism was unclear.One idea was that a positively charged heparin-binding domain in APOA5 interacts with heparan sulfate proteoglycans (HSPGs) on the luminal surface of capillary endothelial cells (ECs), thereby increasing the margination of APOA5-containing TRLs along capillaries and facilitating lipoprotein lipase (LPL)-mediated TG hydrolysis (12,14).That proposal, however, was open to question because the plasma levels of APOA5 are extremely low (15)(16)(17)(18), such that few TRLs have even a single molecule of APOA5 (19,20).Another idea was that APOA5 activates the catalytic activity of LPL (10,11,21), but several studies have been unable to detect any effect of APOA5 on LPL activity (12,14,22).Multiple groups have attempted to gauge the impact of APOA5 deficiency on LPL expression by measuring amounts of LPL activity in the plasma after a bolus of heparin.The results of those studies, both in humans and mice, have been inconclusive; several studies reported that post-heparin LPL levels were low (3)(4)(5)13), whereas others concluded that the LPL levels were normal (6,7,12,23).
A recent report by Yang et al. (30) added to our understanding of APOA5 and ANGPTL3/8 physiology.They found that amounts of LPL inside capillaries of the heart and brown adipose tissue (BAT) were lower in Apoa5 −/− mice than in Apoa5 +/+ mice.That discovery implied that ANGPTL3/8 does more than simply inhibit the catalytic activity of LPL.Specifically, they proposed that ANGPTL3/8 functions to detach LPL from its binding sites within capillaries and that the detachment of LPL from capillaries is suppressed by APOA5 (30).Here, we review data that support that proposal.We will also discuss recent findings showing that carboxyl-terminal sequences in APOA5 are crucial for its ability to bind to ANGPTL3/8 and suppress ANGPTL3/8 activity (31).Finally, we discuss an inhibitory ANGPTL3/8-specific monoclonal antibody (mAb) that functions as an APOA5-mimetic reagent.In mice, the inhibitory mAb, like recombinant APOA5, increases intracapillary LPL levels and sharply reduces plasma TG levels (30).The inhibitory mAbs are likely to be therapeutically useful in humans.In a single-dose human trial, the inhibitory mAb sharply reduced plasma TG levels and led to substantial reductions in both LDL cholesterol (LDL-C), and APOB levels.

APOA5 BINDS TO ANGPTL3/8 AND SUPPRESSES ITS CAPACITY TO INHIBIT LPL CATALYTIC ACTIVITY
ANGPTL3/8 is a physiologic inhibitor of LPL activity in oxidative tissues (25,28,32,33).To screen for ANGPTL3/8-interacting proteins, Chen et al. (22) incubated human serum with ANGPTL3/8-coated beads, and the proteins that bound to the beads were digested with trypsin.Mass spectrometry-based studies revealed enrichment of APOA5 tryptic peptides (22).That finding raised the possibility that APOA5 binds to ANGPTL3/8 and that APOA5 could function to suppress ANGPTL3/8-mediated inhibition of LPL activity.To explore this idea, Chen et al. (22) produced recombinant human APOA5 and human ANGPTL3/8 (22); demonstrated that APOA5 binds to ANGPTL3/8 with high affinity (22); and showed that APOA5 suppresses the ability of ANGPTL3/8 to inhibit LPL activity (22).The binding of APOA5 to ANGPTL3/8 was specific.APOA5 did not bind or inhibit ANGPTL4 [which regulates LPL activity in adipose tissue (34,35)], and published data suggest that APOA5 has little ability to suppress the capacity of ANGPTL3 [an inhibitor of LPL (36) and endothelial lipase (37)] to inhibit LPL catalytic activity (22).
In an independent study, Chen et al. (25) reported that ANGPTL3/8 binds to LPL, implying that ANGPTL3/8 could reduce LPL activity simply by binding to LPL and thereby interfering (in a direct fashion) with LPL's ability to hydrolyze TRL triglycerides.Of note, APOA5 and LPL bind to similar sites on ANGPTL3/8 (38).With these observations in mind, one could propose that the binding of APOA5 to ANGPTL3/8 preserves LPL catalytic activity simply by interfering with ANGPTL3/8 binding to LPL.As it turned out, however, and as discussed later in this review, ANGPTL3/8's impact on intravascular lipolysis is more complicated than simply binding to LPL and interfering with LPL's ability to hydrolyze triglycerides.As explained later, ANGPTL3/8 reduces intracapillary LPL levels, and that effect is suppressed by APOA5.

APOA5 DEFICIENCY RESULTS IN REDUCED AMOUNTS OF LPL WITHIN CAPILLARIES
Recent studies by Yang et al. (30) added to our understanding of the mechanisms by which APOA5 and ANGPTL3/8 affect plasma TG metabolism.They found, by confocal microscopy, that amounts of LPL inside capillaries of oxidative tissues [e.g., heart and brown adipose tissue (BAT)] are lower in Apoa5 −/− mice than in Apoa5 +/+ mice (30).To quantify amounts of LPL on the luminal surface of endothelial cells (ECs), they gave Apoa5 −/− and Apoa5 +/+ mice an intravenous injection of Alexa Fluor-labeled mAbs against LPL, GPIHBP1 (the LPL transporter in ECs), and CD31 (an EC protein) (39).Then, after 10 min, they perfused mice with PBS, perfusion-fixed the tissues, and prepared cryosections for fluorescence microscopy.The amounts of LPL, GPIHBP1, and CD31 in capillaries were assessed by quantifying Alexa Fluor signals in large numbers of capillary segments (n = 323-1870 segments/tissue/mouse in four independent experiments).These studies revealed that amounts of LPL inside capillaries, relative to GPIHBP1 or CD31, were significantly reduced in BAT and heart capillaries of Apoa5 −/− mice (Fig. 1).Consistent with these observations, they found reduced amounts of LPL mass and activity in the post-heparin plasma of Apoa5 −/− mice.Also, the margination of TRLs along the luminal surface of capillaries [a process that depends on intracapillary LPL (40,41)] was lower in Apoa5 −/− mice than in Apoa5 +/+ mice.These observations led Yang et al. (30) to propose that the in vivo effect of ANGPTL3/8 is more complex than simply binding to LPL and blocking LPL's TG hydrolase activity.They proposed that ANGPTL3/8 detaches LPL from the luminal surface of capillaries and that the detachment of LPL is increased in the setting of APOA5 deficiency (where ANGPTL3/8 activity is unsuppressed).(44) demonstrated that ANGPTL4 binds to sequences surrounding LPL's catalytic pocket and that this binding event initiates the unfolding of LPL's native conformation, explaining the irreversible enzyme inactivation.Based on the ANGPTL4 precedent, Yang et al. (30) speculated that ANGPTL3/8 might unfold LPL conformation and that the ANGPTL3/8-mediated unfolding might account for both the loss of LPL catalytic activity and the detachment of LPL from the surface of capillaries.We emphasize, however, that this was merely speculation; the molecular mechanism underlying ANGPTL3/8 activity requires rigorous testing under a variety of experimental conditions.

ANGPTL3/8 DETACHES LPL FROM THE SURFACE OF CULTURED CELLS, AND THAT DETACHMENT IS BLOCKED BY APOA5 AND BY AN INHIBITORY ANGPTL3/8-SPECIFIC mAb
Yang et al. (45) tested whether recombinant ANGPTL3/8 was capable of detaching LPL from the surface of cultured cells, and if so, whether the detachment of LPL could be blocked by APOA5.First, they loaded cell-surface HSPGs of CHO-K1 cells with human LPL by incubating the cells with recombinant LPL.The cells were then washed and incubated with cell culture medium alone, ANGPTL3/8, or ANGPTL3/8 in the presence of either recombinant APOA5 or an inhibitory ANGPTL3/8-specific mAb (IBA490).IBA490 binds to an epitope in ANGPTL3/8 that overlaps with the APOA5 binding site (38).The surface of cells was then stained with an Alexa Fluor 555-labeled mAb against human LPL (5D2) and an Alexa Fluor 488-labeled wheat germ agglutinin (WGA, which binds to cell-surface HSPGs).Amounts of LPL on the surface of cells, as judged by 5D2 binding, were reduced in the cells that had been incubated with ANGPTL3/8 alone (Fig. 2).In cells that had been incubated with ANGPTL3/8 + APOA5 or ANGPTL3/8 + IBA490, amounts of LPL on the cell surface were not reduced.These findings provided a very plausible explanation for the reduced amounts of LPL along the luminal surface of capillaries in Apoa5 −/− mice.impact of both APOA5 and IBA490 on intracapillary LPL levels in oxidative tissues in Apoa5 −/− mice.To explore the effects of APOA5, mice were given an intravenous injection of recombinant APOA5 or PBS alone.After 4 h, the mice were given an intravenous injection of Alexa Fluor-labeled mAbs against LPL, GPIHBP1, and CD31.After 10 min, cryosections of heart and BAT were prepared for confocal fluorescence microscopy.Amounts of LPL along the luminal surface of capillaries, relative to GPIHBP1 or CD31, were significantly increased in the Apoa5 −/− mice that had received recombinant APOA5 (30) (Fig. 3).Consistent with that finding, the plasma TG levels in the mice that had been given APOA5 fell dramatically (from 1116 to 87 mg/dl) (30) (Fig. 4).
To assess the impact of mAb IBA490 on TG metabolism, Apoa5 −/− mice were given a subcutaneous injection of IBA490 or an irrelevant control IgG (30).After 24 h, the mice were given an intravenous injection of Alexa Fluor-labeled mAbs against LPL, GPIHBP1, and CD31.After 10 min, the vasculature was perfused and fixed, and tissue cryosections were prepared for microscopy.Amounts of intracapillary LPL in the heart and BAT of Apoa5 −/− mice, relative to GPIHBP1 or CD31, were increased after IBA490 treatment (Fig. 5), and the plasma TG levels fell dramatically from 1291 to 52 mg/dl (30) (Fig. 4).The low plasma TG levels after IBA490 were sustained for three days (30).
In the microscopy studies designed to assess the impact of APOA5 and IBA490 on intracapillary LPL levels, the fluorescent signals for the mAbs against LPL, GPIHBP1, and CD31 were quantified in large numbers of capillaries (n = 381-2020 capillary segments/tissue/mouse in three independent experiments) (30).The increased intracapillary LPL levels after APOA5 and IBA490 were visibly apparent in the confocal micrographs and were confirmed by quantification of the fluorescent signals.
For example, the LPL:GPIHBP1 and LPL:CD31 fluorescent intensity ratios in heart capillaries were significantly elevated in Apoa5 −/− mice that had been treated with APOA5 and IBA490 (30).
The ability of APOA5 and IBA490 to increase intracapillary LPL levels was not confined to Apoa5 −/ − mice.APOA5 and IBA490 infusions also increased intracapillary LPL levels in Apoa5 +/+ mice, as judged by confocal microscopy-based measurements of LPL:GPIHBP1 fluorescent intensity ratios (30).The ability of IBA490 to influence intracapillary LPL levels in Apoa5 +/+ mice was strongly supported by a study by Yang et al. (46) on levels of LPL inside heart capillaries during fasting and refeeding.More than three decades ago, Kuwajima et al. (47) demonstrated that LPL expression in the heart is high during fasting and low after refeeding, but the mechanism was unknown.We now know that refeeding triggers a ∼9-fold increase in the plasma levels of ANGPTL3/8 (25,31).Yang et al. (46) showed, with immunofluorescence confocal microscopy, that amounts of LPL inside heart capillaries, relative to GPIHBP1 or CD31, are high in fasted Apoa5 +/+ mice but fall by 50%-60% after refeeding.They also showed that the decrease in intracapillary LPL levels after refeeding can be abolished by inhibiting ANGPTL3/8 activity with mAb IBA490 (46).
We have not assessed the impact of APOA5 deficiency on the background of ANGPTL8 deficiency.Because APOA5 functions to suppress ANGPTL3/8 activity, our Fig. 3. Confocal micrographs depicting amounts of LPL inside heart capillaries of Apoa5 −/− and Apoa5 +/+ mice treated with recombinant APOA5 or phosphate-buffered saline (PBS) alone.Apoa5 −/− and Apoa5 +/+ mice were given an intravenous injection of APOA5 (10 mg/kg) or PBS.Four h later, the amount of LPL inside heart capillaries in mice was measured by confocal microscopy, as described in Fig. 1.Intracapillary LPL levels in the heart were low in Apoa5 −/− mice and were increased by recombinant APOA5 (30).Scale bars, 20 μm.
APOA5 deficiency causes low intracapillarly LPL levels expectation is that APOA5 deficiency would have little impact on LPL levels in capillaries of heart and brown adipose tissue in Angptl8-deficient mice.However, interpreting such a model could be complicated by the absence of the ANGPTL4/8 complex in adipose tissue.An absence of the ANGPTL4/8 complex in adipose tissue could result in reduced amounts of intracapillary LPL in adipose tissue (29).Confocal micrographs depicting amounts of LPL inside heart capillaries of Apoa5 +/+ and Apoa5 −/− mice treated with either mAb IBA490 or an irrelevant control IgG.Apoa5 −/− and Apoa5 +/+ mice were given a subcutaneous injection of IBA490 (10 mg/kg) or an irrelevant control IgG (10 mg/kg).After 24 h, amounts of intracapillary LPL in mice were assessed by fluorescent microscopy, as described in Fig. 1.Intracapillary LPL levels in the heart were low in Apoa5 −/− mice but were increased by mAb IBA490 (30).Scale bars, 20 μm.

KEY POINTS
• APOA5 binds to the ANGPTL3/8 complex and suppresses its ability to inhibit LPL's TG hydrolase activity.(48,49).Those clinical findings inspired Chen, Yang, and coworkers to test whether the C-terminal sequences in human APOA5 are required for APOA5's ability to suppress ANGPTL3/8 activity (31).
In vitro biochemical studies revealed that wild-type (WT) human APOA5, but not a mutant human APOA5 lacking 35 C-terminal amino acids (APOA5Δ35), blocked the ability of ANGPTL3/8 to inhibit LPL's TG hydrolase activity.A truncated human APOA5 lacking 92 carboxyl-terminal residues also failed to suppress ANGPTL3/8 activity (31).To pursue these observations, Chen, Yang, et al. (31) introduced the "APOA5Δ35 frameshift mutation" into an expression vector for mouse APOA5 and then expressed and purified a truncated mouse APOA5 (APOA5Δ40).[The truncation mutation in mouse APOA5 results in the deletion of 40 residues rather than 35 (owing to a 5-residue extension at the C-terminus of mouse APOA5).]Surface plasmon resonance (SPR) studies revealed that WT mouse APOA5 bound to ANGPTL3/8 with high affinity (K D = 0.53 nM), whereas there was no measurable binding of mouse APOA5Δ40 to ANGPTL3/8 (Fig. 6) (31).Consistent with that finding, ANGPTL3/8-mediated detachment of LPL from the surface of cultured cells was suppressed by WT mouse APOA5 (and by mAb IBA490) but not by APOA5Δ40 (Fig. 2).The cell culture experiments suggested that APOA5Δ40 would have no ability to influence plasma TG metabolism in Apoa5 −/− mice.Indeed, WT-APOA5, but not APOA5Δ40, sharply reduced plasma TG levels in Apoa5 −/− mice (Fig. 4).Also, WT-APOA5, but not APOA5Δ40, increased intracapillary LPL levels in the heart of Apoa5 −/− mice (Fig. 7A).Fig. 6.Testing the binding of mouse WT-APOA5 or APOA5Δ40 to ANGPTL3/8 by surface plasmon resonance (SPR).A CM4 sensor chip (coupled with a rabbit anti-mouse IgG) was primed with ANGPTL3/8-specific mAb 1G12 (which binds to the C-terminal fibrinogen-like domain) or mAb IBA490 (which binds to an epitope that overlaps with the APOA5 binding site).After an injection of 25 nM ANGPTL3/8, comparable amounts of ANGPTL3/8 were immobilized on the chip.The binding affinity of APOA5 proteins to immobilized ANGPTL3/8 was measured with injections of 1:2 serial dilutions of 0.5-8 nM WT-APOA5 or APOA5Δ40 (arrows).The binding of WT-APOA5 to 1G12-immobolized ANGPTL3/8 fits a simple bimolecular interaction model and yielded a dissociation constant (K D ) of 0.53 nM, whereas the binding of APOA5Δ40 to 1G12-immobolized ANGPTL3/8 and the binding of WT-APOA5 to IBA490-captured ANGPTL3/8 were virtually undetectable.Reproduced with permission from Chen, Yang, et al. (31).

APOA5 deficiency causes low intracapillarly LPL levels AN ANTIBODY AGAINST THE LAST 26 RESIDUES OF MOUSE APOA5 BLOCKS THE ABILITY OF APOA5 TO SUPPRESS ANGPTL3/8 ACTIVITY IN VITRO AND IN VIVO
To further explore the idea that C-terminal sequences in APOA5 are crucial for regulating ANGPTL3/8 activity, Chen, Yang, and coworkers generated a synthetic peptide corresponding to the last 26 residues of mouse APOA5 and then made a rabbit polyclonal antibody, CT-APOA5 pAb, against the peptide (31).CT-APOA5 pAb bound to WT-mouse APOA5 but not to APOA5Δ40, as judged by western blot studies and immunohistochemistry studies on WT mouse liver (31).In vitro biochemical studies revealed that CT-APOA5 pAb abolished the ability of WT mouse APOA5 to suppress ANGPTL3/8-mediated inhibition of LPL catalytic activity (31).Also, CT-APOA5 pAb inhibited the Fig. 7. Confocal microscopy studies to assess the relevance of carboxyl-terminal APOA5 sequences for APOA5's ability to regulate intracapillary LPL levels.A: Apoa5 −/− mice were given an injection of wild-type mouse APOA5 (WT-APOA5; 0.5 nmole/mouse), a truncated APOA5 lacking the last 40 residues of the protein (APOA5Δ40; 0.5 nmole/mouse), or vehicle (PBS) alone.After 4 h, the mice were given an intravenous injection of Alexa Fluor-labeled mAbs against LPL, GPIHBP1, and CD31.After 10 min, heart cryosections were prepared; and amounts of LPL, GPIHBP1, and CD31 on the luminal surface of capillaries were assessed by confocal microscopy and by quantifying LPL, GPIHBP1, and CD31 fluorescence intensities (31).WT-APOA5, but not APOA5Δ40, increased amounts of LPL in heart capillaries of Apoa5 −/− mice.Scale bars, 20 μm.B: fasted wild-type mice were given an intravenous injection of CT-APOA5 pAb (1.5 mg/mouse) or a nonimmune rabbit IgG (control IgG; 1.5 mg/mouse).After allowing the mice to refeed a chow diet for 6 h, the mice were given an intravenous injection of Alexa Fluor-labeled mAbs against LPL, GPIHBP1, and CD31.After 10 min, the tissues were fixed, and fluorescence intensities were imaged and quantified by confocal microscopy (31).The mice that had been treated with CT-APOA5 had reduced amounts of LPL in heart capillaries, consistent with the ability of CT-APOA5 pAb to neutralize the activity of APOA5.Scale bars, 50 μm.Reproduced with permission from Chen, Yang, et al. (31).ability of APOA5 to suppress ANGPTL3/8-mediated detachment of LPL from the surface of cultured cells (31).When CT-APOA5 pAb was infused into Apoa5 +/+ mice, plasma TG levels increased from 96 to 304 mg/dl within 6 h, whereas an infusion of nonimmune rabbit IgG had no effect on plasma TG levels (Fig. 8) (31).Consistent with that observation, CT-APOA5 pAb, but not the control rabbit IgG, reduced amounts of LPL inside heart capillaries of Apoa5 +/+ mice (Fig. 7B) (31).
The studies by Chen, Yang, and coworkers (31) revealed that C-terminal APOA5 sequences are crucial for the suppression of ANGPTL3/8 activity, but the identities of specific APOA5 amino acids that are important for the binding of APOA5 to ANGPTL3/8 have not yet been defined.Interestingly, AlphaFold3 predicts that the C-terminus of mouse APOA5 (S330-S358) contains an α-helix with hydrophobic residues (L334, L337, L341, L344, I348, L352) on one surface of the helix.These residues are conserved in human and rat APOA5 (31,50).While the confidence level for the AlphaFold3 structural predictions for APOA5's C-terminal region was low, we nevertheless believe that it will be important, in future studies, to test whether the stretch of hydrophobic residues is crucial for APOA5's ability to bind to ANGPTL3/8 and suppress its biological activity.

IBA490 AND APOA5 HAVE THE SAME EFFECTS
ON PLASMA TG METABOLISM Recombinant APOA5 and IBA490 both suppress the ability of ANGPTL3/8 to inhibit LPL catalytic activity in vitro; both block ANGPTL3/8-mediated LPL detachment from cultured cells; both increase intracapillary LPL levels in heart and BAT; and both sharply reduce plasma TG levels in Apoa5 −/− mice (30).The fact that APOA5 and IBA490 have the same effects on intravascular lipolysis is not surprising.Earlier hydrogen-deuterium exchange/mass spectrometry studies revealed that the binding site on ANGPTL3/8 for a human ANGPTL3/8-specific mAb (5G11) overlaps with the binding site for APOA5 (38).
[The variable domain of 5G11 and IBA490 are identical; IBA490 differs from 5G11 only by having a mouse Fc domain.]Because the binding sites for APOA5 and mAbs 5G11 and IBA490 on ANGPTL3/8 overlap and because APOA5 and the mAbs have the same effects on TG metabolism, 5G11 and IBA490 can be considered APOA5-mimetic reagents.

PROSPECTS FOR TREATING HYPERLIPIDEMIC PATIENTS WITH AN INHIBITORY ANGPTL3/8 mAb
The recent studies on APOA5 deficiency by Yang and coworkers (30) suggested that an inhibitory ANGPTL3/8 mAb (5G11) could be effective for treating patients with loss-of-function APOA5 mutations.Effective therapies for APOA5 deficiency are important.Han Chinese populations from Southeast Asia harbor a pathogenic APOA5 missense variant (p.G185C) that has been linked to elevated plasma TG levels (5) and an increased risk for coronary artery disease (9).That variant has an allele frequency of 7%, implying that there could be ∼100 million carriers in China alone.
The clinical utility of the inhibitory ANGPTL3/8 mAb could extend beyond patients with an inherited Fig. 8. Testing the impact of a polyclonal antibody against the C-terminal 26 residues of APOA5 (CT-APOA5 pAb) on plasma TG levels in Apoa5 +/+ mice.Fasted Apoa5 +/+ mice (n = 7/group) were given an intravenous injection of CT-APOA5 pAb (1.5 mg/mouse) or a nonimmune rabbit IgG (control IgG; 1.5 mg/mouse) and then allowed to refeed a chow-diet.Plasma TG levels (mean ± SD) were measured at baseline and 6 h after the antibody injections.CT-APOA5 pAb increased plasma TG levels in Apoa5 +/+ mice from 96 ± 22 mg/dl to 304 ± 74 mg/dl, whereas TG levels in mice that received control IgG remained low.Figures were created from data reported by Chen, Yang, et al. (31).deficiency of APOA5.In wild-type mice, the mAb IBA490 resulted in higher intracapillary LPL levels and lower plasma TG levels (30).Also, increased expression of APOA5 (which functions to suppress ANGPTL3/8 activity) reduces plasma TG levels in wild-type mice (1,10,51).
The ability of mAb 5G11 to reduce plasma lipid levels was tested in a 28-day, randomized, double-blind, placebo-controlled trial of 48 human subjects with mixed hyperlipidemia (52).At the highest dose, 5G11 reduced plasma TG levels by 70%, remnant cholesterol levels by 61%, LDL-C levels by 36%, and APOB levels by 31% while increasing HDL cholesterol levels by 26% (52).Plasma TG lowering persisted for two weeks after the 5G11 infusion (52).Longer-term testing of 5G11 in different patient populations is needed, but it seems likely that mAb 5G11 will prove useful for treating a wide range of hyperlipidemia patients.The fact that 5G11 reduces plasma levels of both APOB and LDL-C raises the possibility that it could prove useful for reducing the risk of coronary heart disease.CONCLUSIONS Now, two decades after the discovery of APOA5 and the discovery of severe hypertriglyceridemia in Apoa5 −/− mice (1), the molecular physiology of APOA5 has come into focus.APOA5 binds to ANGPTL3/8 and suppresses its ability to inhibit LPL-mediated processing of TRLs (Fig. 9A).In the setting of APOA5 deficiency, ANGPTL3/8 activity is unbridled, resulting in reduced inhibition of LPL catalytic activity in vitro and reduced amounts of intracapillary LPL in vivo (Fig. 9B).The inhibitory ANGPTL3/8-specific mAbs (IBA490, 5G11) are APOA5-mimetic agents; they suppress the ability of ANGPTL3/8 to inhibit LPL catalytic activity and to reduce intracapillary LPL levels (Fig. 9C).A study of 48 human subjects with mixed hyperlipidemia revealed that mAb 5G11 reduces plasma lipid levels of TGs, LDL-C, and APOB very effectively.

In 2001 ,
Pennacchio et al. (1) uncovered a new gene, APOA5, by comparative sequencing of the human and mouse APOA1/APOC3/APOA4 gene cluster.Similarities between the structures of APOA5 and other apolipoproteins raised the possibility that APOA5 could play a role in lipoprotein metabolism.To explore that possibility, Pennacchio et al. (1) created Apoa5-deficient mice (Apoa5 −/− The notion that ANGPTL3/8 could both inactivate LPL activity and detach LPL from capillaries was plausible.Earlier, Mysling et al. (42) proved, with hydrogen-deuterium exchange/mass spectrometry studies, that ANGPTL4 regulates LPL activity by catalyzing the unfolding of LPL's hydrolase domain (resulting in irreversible loss of LPL catalytic activity).Follow-up studies by Leth-Espensen et al. (43) and Kumari et al.

INHIBITING ANGPTL3/ 8
ACTIVITY WITH EITHER APOA5 OR mAb IBA490 INCREASES INTRACAPILLARY LPL LEVELS IN APOA5 −/− MICE AND NORMALIZES PLASMA TG LEVELS The ability of APOA5 and IBA490 to suppress ANGPTL3/8-mediated detachment of LPL from cultured cells prompted Yang et al. (30) to test the

Fig. 1 .
Fig. 1.Confocal micrographs depicting amounts of LPL inside heart capillaries of Apoa5 +/+ and Apoa5 −/− mice.To assess intracapillary LPL levels, Apoa5 −/− and Apoa5 +/+ mice were given an intravenous injection of Alexa Fluor-labeled monoclonal antibodies (mAbs) against LPL, GPIHBP1, and CD31.After 10 min, cryosections of the heart were prepared for microscopy.Amounts of LPL on the luminal surface of capillaries, relative to amounts of GPIHBP1 and CD31, were assessed by quantifying fluorescent intensities.Fluorescence intensity ratio data are found in the paper by Yang et al. (30).Scale bars, 20 μm.

Fig. 9 .
Fig.9.Schematic depictions of intracapillary triglyceride metabolism in wild-type mice, Apoa5 −/− mice, and Apoa5 −/− mice after an infusion of the inhibitory ANGPTL3/8-specific mAb IBA490.A: In wild-type mice, APOA5 binds to ANGPTL3/8 and suppresses its ability to inactivate LPL and blocks its ability to detach LPL from the luminal surface of capillary endothelial cells, thereby preserving robust lipolytic processing of triglyceride-rich lipoproteins (TRLs).B: In Apoa5 −/− mice, ANGPTL3/8 activity is increased because of the absence of APOA5.The increased ANGPTL3/8 activity inactivates LPL and detaches it from the surface of capillaries, thereby impeding the lipolytic processing of TRLs.C: In Apoa5 −/− mice, mAb IBA490 mimics the activity of APOA5.IBA490 binds to ANGPTL3/8 and suppresses ANGPTL3/8 activity, thereby increasing intracapillary LPL levels and augmenting the efficiency of TRL processing.